High performance liquid chromatography (HPLC) is a process by which one or more compounds from a chemical mixture may be separated and identified. A transport liquid, for example a solvent, is pumped under high pressure through a column of packing medium, and a sample of the chemical mixture to be analyzed is injected into the column. As the sample passes through the column with the liquid, the different compounds, each one having a different affinity for the packing medium, move through the column at different speeds. Those compounds having greater affinity for the packing material move more slowly through the column than those having less affinity, and this speed differential results in the compounds being separated from one another as they pass through the column.
The transport liquid with the separated compounds exits the column and passes through a detector, which identifies the molecules, for example by spectrophotometric absorbance measurements. A two dimensional plot of the detector measurements against elution time or volume, known as a chromatogram, may be made, and from the chromatogram the compounds may be identified.
For each compound, the chromatogram displays a separate curve or “peak”. Effective separation of the compounds by the column is advantageous because it provides for measurements yielding well defined peaks having sharp maxima inflection points and narrow base widths, allowing excellent resolution and reliable identification of the mixture constituents. Broad peaks, caused by poor column performance, are undesirable as they may allow minor components of the mixture to be masked by major components and go unidentified.
The uniformity of the packing medium within the column has a significant effect on column performance. It is desired that the particles comprising the packing medium be perfectly arranged and completely homogeneous so that the transport liquid and the sample mixture move at uniform rates through the column. Areas of loose packing medium create channels causing locally increased flow rates while areas that are partially plugged due to particle aggregation create eddies that retard the flow. Such local variations in the flow rate caused by non-uniform packing medium result in transport liquid mixing that degrades the column performance resulting in broadening of the peaks and a concomitant decrease the resolving capability of the HPLC apparatus.
Columns for HPLC are packed with packing media comprising, for example, silane derivatized silica spheres having a diameter less than 20 microns. Packing is performed at high pressures, typically between 7,000 and 9,000 psi, three to four times the pressure at which the column will operate. Under such high pressures, the columns, made from thick wall steel tubing, will expand radially as much as five packing particle diameters depending upon the ratio of wall thickness to inner diameter. Upon completion of packing, the pressure is removed and the remaining column components are installed. Removal of the pressure allows the column to relax and contract radially to its nominal diameter. This places the packing media under compression, and the hydraulically oriented layers of packing media buckle and shift in response. The buckling action disrupts the uniformity of the layers which adversely affects column performance. There is clearly a need for an HPLC column which does not suffer disruption of the hydraulic orientation of the packing media upon removal of the pressure imposed during packing.